In the optical method for the determination of glucose, hexokinase (HK) catalyzes the phosphorylation of glucose by adenosine triphosphate (ATP) as follows: EQU Glucose + ATP .sup.Hexokinase .fwdarw. Glucose-6-Phosphate + Adenosine Diphosphate (ADP)
glucose-6-phosphate (G-6-P) is oxidized in the presence of nicotinamide adenine dinucleotide (NAD) by glucose-6-phosphate dehydrogenase (G-6-PDH): EQU G-6-P + NAD.sup.+ .sup.G-6-PDH .fwdarw. 6-Phosphogluconate + NADH + H.sup.+
the reduction of NAD to NADH (reduced form of NAD) at 340 nanometers (nm) is a quantitative measure of the amount of glucose present.
In the creatine phosphokinase (CPK) assay method of Rosalki, s. b., Journal of Laboratory and Clinical Medicine, 69; 696 1967), CPK catalyzes the reversible formation of adenosine triphosphate (ATP) and creatine from adenosine diphosphate (ADP) and creatine phosphate (CPO.sub.4) according to the equation: EQU ADP + Creatine Phosphate .sup.CPK .fwdarw. ATP + Creatine
The ATP formed in the CPK mediated reaction is used to phosphorylate glucose in the presence of hexokinase (HK) producing glucose-6-phosphate. As glucose-6-phosphate is formed, ADP is generated keeping its concentration at a constant level. EQU Glucose + ATP .sup.Hexokinase .fwdarw. Glucose-6-Phosphate + ADP
the glucose-6-phosphate formed by the hexokinase reaction is then oxidized by the enzyme glucose-6-phosphate dehydrogenase (G-6-PDH) with simultaneous reduction of NAD. EQU G-6-P +NAD.sup.+ .sup.G-6-PDH.fwdarw. 6-Phosphogluconate+ NADH + H.sup.+
the reduction of NAD to NADH is followed spectrophotometrically by observing the resulting increase in absorbance at 340 nm. For each mole of phosphate transferred by the CPK, one mole of NADH is formed. Thus the rate of absorbance change is directly proportional to the CPK activity present in the sample.
Lactic dehydrogenase (LDH) (L-Lactate:NAD oxidoreductase catalyzes the following reaction: EQU L-(+):Lactate + NAD.sup.+ .sup.LDH .revreaction. Pyruvate + NADH + H.sup.+
in assaying for serum constituents other than LDH, any LDH present may cause interference due to the presence of either lactate or pyruvate in the sample, thus causing errors in the assay of glucose or CPK or in any assay system where NAD is reduced to NADH. Any NADH generated is subject to reaction with pyruvate in the presence of LDH, thus causing errors in the assay of glucose or CPK, for example. EQU H.sup.+ + NADH + Pyruvate .sup.LDH .fwdarw. Lactate + NAD.sup.+
this interference may be overcome or avoided by the use of NADP as follows: EQU 1. Creatine Phosphate (CPO.sub.4) + ADP .sup.CPK .fwdarw. Creatine + ATP EQU 2. atp + glucose .sup.Hexokinase .fwdarw. Glucose-6-Phosphate (G-6-P) + ADP EQU 3. g-6-p + nad phosphate (NADP).sup.+ .sup.G-6-PDH .fwdarw. 6-Phosphogluconate + NADPH + H.sup.+
nadp and NADPH do not react with LDH present in common biological samples; however, the use of NADP in the enzymatic determination of either CPK or glucose increases the cost of the reagent and reduces the dynamic range of the method. The use of NAD in place of NADH results in reduced costs of reagent and increased dynamic range but permits undesirable reactions and the possibility of error as hereinbefore described.